Compelling evidence links the development of muscle insulin resistance to fatty acid (FA) oversupply, but there is still uncertainty regarding which specific FA metabolite(s) and regulatory pathways are directly responsible for mediating insulin desensitization. Our current knowledge is based largely on a candidate molecule approach in which the selection of potentially relevant lipid metabolites relies heavily on implicit biases or availability of specific assays. To overcome this obstacle, we have employed mass spectroscopy-based metabolic profiling to comprehensively evaluate multiple lipid-derived metabolites in muscle from rats made insulin resistant by a high fat diet compared to controls. This unbiased "metabolomics" approach led to our discoveries that insulin resistant rats exhibit marked intramuscular accumulation of the ketone, beta-hydroxybutyrate (betaHB), at least a portion of this accumulated ketone was synthesized directly in the muscle tissue, and that genetic manipulations that restored insulin sensitivity corresponded with a 55% decrease in muscle (HB levels. These exciting findings implicate muscle ketone dysregulation as a causal factor in the etiology of lipid-induced insulin resistance. Previous research supports an inverse correlation between ketogenesis and glucose intolerance, but no consideration has been given to the possibility that dysregulated metabolism of ketones within muscle tissue could contribute to insulin resistant states. We hypothesize that chronic high fat feeding and/or overnutrition imposes a state of persistent and abnormal ketogenesis in skeletal muscle, which in turn plays a direct role in causing maladaptive changes in glucose handling and insulin sensitivity. We propose to test these hypotheses with the following specific aims: 1) To determine whether ketone dysregulation is a common feature in multiple animal models of insulin resistance. This will be done by profiling gene expression and metabolic markers of ketone metabolism in rodent models of obesity and diabetes. 2) To determine whether perturbing ketone metabolism (by FA overexposure or by adenovirus-mediated delivery of ketogenic genes), disrupts glucose handling and insulin signaling in isolated muscles and/or muscle cells in culture. 3) To determine whether adenovirus-mediated overexpression of genes that suppress ketogenesis and/or enhance ketone degradation in muscle can reverse diet-induced insulin resistance in vivo. The results from these studies are expected to yield important new insights into the mechanism of FA-induced insulin resistance in skeletal muscle, with potential therapeutic implications for treatment of type 2 diabetes.